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. 2016 Mar 2;11(3):e0150037.
doi: 10.1371/journal.pone.0150037. eCollection 2016.

Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors

Pedro Perdigão  1   2   3   4 , Thomas Gaj  2   3   4 , Mariana Santa-Marta  1 , Carlos F Barbas 3rd  2   3   4 , Joao Goncalves  1
Affiliations

Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors

Pedro Perdigão et al. PLoS One. .

Abstract

The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. TALE transcription factors (TALE-TFs) designed to target the HIV-1 LTR promoter.
(A) Schematic representation of the TALE transcription activator (TLT) binding sites within the HIV-1 long terminal repeat (LTR) promoter relative to the transcriptional start site (TSS) and main endogenous transcription factor binding sites. (B) (Left) Cartoon illustrating the structure of a TALE-TF, adapted from [109]. TALE repeats are colored cyan and purple, DNA shown as grey sticks. (Right) Schematic representation of the TLT expression construct used in this study. CMV indicates the cytomegalovirus promoter, TALE repeats are shown as individual bars (16 repeats total), VP64 denotes the tetrameric repeat of the herpes simplex virus VP16 transactivation domain, NLS stands for the nuclear localization signal derived from the simian virus (SV40) and HA indicates the hemagglutinin A tag. (C) Schematic representation of the luciferase reporter system containing four direct repeats of the TALE target sites for each TALE activator. Each TALE target site is shown. (D) (Top) Fold-activation of luciferase expression after co-transfection of TALE-TFs with luciferase reporter plasmid into HEK293T cells. Luciferase expression was normalized to cells transfected with reporter plasmid only. Renilla luciferase expression was used to normalize for transfection efficiency and cell number. Error bars indicate standard deviation of one experiment with three transfection replicates (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; t-test sample vs control (4x TALE binding site vector only)). (Bottom) Western blot of lysate from HEK293T cells transfected with TALE-TFs. Samples were taken 48 h after transfection and probed with horseradish peroxidase-conjugated anti-HA and anti-β-actin (loading control) antibodies. Empty indicates lysate from HEK293T cells transfected with empty pcDNA vector only.
Fig 2
Fig 2. TALE-TF-mediated gene activation from the HIV-1 LTR promoter.
(Top) Schematic representation of the luciferase reporter system used to evaluate TALE-TF activity from the HIV-1 LTR promoter. The U3 and R regions of the HIV LTR were placed upstream of the luciferase reporter. (Bottom) Fold-activation of luciferase expression in HEK293T cells co-transfected with reporter plasmid and TALE-TF or Tat expression vectors. Luciferase expression was normalized to cells transfected with reporter plasmid only. Error bars indicate standard deviation of one experiment with three transfection replicates (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; t-test sample vs. control (TLTNT)).
Fig 3
Fig 3. Reactivation of latent HIV-1 expression by TALE-TFs in multiple cell line models of HIV-1 latency.
(A) (Top) Schematic representation of the HIV-1 proviral genome present in J-Lat cells. Full-length HIV-1 was derived from the molecular clone pNL4-3-ΔEnv-GFP and expresses a GFP gene from the LTR promoter. Structural viral genes are shown in black, auxiliary genes are shown in grey. The nef and env genes were inactivated to force a single infection cycle. (Bottom) Percentage of GFP positive J-Lat 10.6 cells after nucleofection with TALE-TF and Tat expression plasmids, or treatment with TNF-α (10 ng/μL). GFP positive cells were measured by flow cytometry 48 h after nucleofection. “J-Lat” indicates non-transfected J-Lat 10.6 cells. “Mock” indicates cells transfected with an empty pcDNA backbone. Error bars indicate standard deviation of three independent experiments (n = 3). (B) Percentage of GFP positive J-Lat 6.3, 8.4 and 9.2 cells after nucleofection with TLT5-8 and Tat expression plasmids, or treatment with TNF-α (10 ng/μL). GFP positive cells were measured by flow cytometry 48 h after nucleofection. “J-Lat” indicates non-nucleofected cells. Error bars indicate standard deviation (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; t-test sample vs control (J-Lat)).
Fig 4
Fig 4. Enhanced reactivation of latent HIV-1 expression by combining TALE-TFs with a histone deacetylase inhibitor.
Percentage of GFP-positive J-Lat 10.6 and 6.3 cells after nucleofection with TLT5-8 expression plasmids and treatment with increasing concentrations of SAHA or DMSO (0.1%) for 24 h. GFP-positive cells were measured by flow cytometry 48 h after nucleofection. Error bars indicate standard error of the mean of three independent experiments (n = 3; *p < 0.05; **p < 0.01; t-test).
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References

    1. Hammer SM, Katzenstein DA, Hughes MD, Gundacker H, Schooley RT, Haubrich RH, et al. A Trial Comparing Nucleoside Monotherapy with Combination Therapy in HIV-Infected Adults with CD4 Cell Counts from 200 to 500 per Cubic Millimeter. New England Journal of Medicine. 1996. pp. 1081–1090. - PubMed
    1. Perelson AS, Essunger P, Cao Y, Vesanen M, Hurley A, Saksela K, et al. Decay characteristics of HIV-1-infected compartments during combination therapy. Nature. 1997;387: 188–191. - PubMed
    1. Gulick RM, Mellors JW, Havlir D, Eron JJ, Gonzalez C, McMahon D, et al. Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. The New England journal of medicine. 1997. - PubMed
    1. Chun TW, Davey RT, Engel D, Lane HC, Fauci AS. Re-emergence of HIV after stopping therapy. Nature. 1999;401: 874–875. - PubMed
    1. Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci U S A. 1997;94: 13193–13197. - PMC - PubMed

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